From a SWAN to Chopra-Amiel-Gordon Syndrome and the emergence of “-like” syndromes, CNTNAP2 etc

 

SWAN (Syndrome Without A Name)

 Today’s post is complicated, it is aimed at people who:

·        are interested in genetic testing for autism 

·        are affected by miss-expression of the genes:-

·        ANKRD17

·        TCF4

·        CNTNAP2

·        NRXN1

 

It is one of those posts that could go on forever; the more you dig, the more you uncover and you wonder why other people (salaried researchers) are not doing this.

Today’s post is mainly about a gene called ANKRD17, but it does highlight more general principles about those genetic testing results, some parents strive to obtain.  It does look at downstream effects of Pitt Hopkins Syndrome and “Pitt Hopkins-like Syndrome”, which likely merge into mainstream autism.

Many single gene autisms have already been identified, some have names and some are still SWANs (Syndromes Without A Name). Some syndromes have long been identified,  but their biological basis had not been identified.  From last year, loss of function of the gene ANKRD17 became Chopra-Amiel-Gordon Syndrome.  

Our reader Mary’s whole exome sequencing (WEG) from a few years ago, for her daughter, has now been flagged up as carrying a mutation leading to Chopra-Amiel-Gordon Syndrome.

In effect, the mutation in ANKRD17 went from be of no confirmed relevance to autism, to being causal, thanks to Dr Chopra and her pals.

This highlights the weakness in the interpretation of genetic testing.  Any benchmark list of autism genes is just a work in progress; your mutation may not yet be there.

Another gene recently queried by a reader was CNTNAP2, this turns out to a key DEG (differentially expressed gene) of a syndrome with its own name, Pitt Hopkins Syndrome, caused by reduced expression of TCF4 (Transcription Factor 4).  Reduced expression of TCF4 has very many effects, but one effect is to reduce expression of CNTNAP2.

In lay-speak, lack of TCF4 causes a cascade of effects, one of which is on the expression of CNTAP2.  We see that people with a CNTAP2 mutation share many of the features of people having a TCF4 mutation.  So, of all the many effects caused by TCF4, those along the TCF4-CNTAP2 pathway should be focused on.  The mutation in CNTNAP2, quite rationally, is now called Pitt Hopkin-like Syndrome-1.  There is also a Pitt Hopkin-like Syndrome-2 which is caused by a mutation in NRXN1 (neurexin 1). 

 

https://royalsocietypublishing.org/doi/pdf/10.1098/rsob.210091

 

In mammals, the neurexins are encoded by three NRXN genes (NRXN1-3), each of which has both an upstream promoter that is used to generate the α-neurexins, and a downstream promoter that is used to generate the shorter β-neurexins [13,15].

 

α-neurexins are composed of six large extracellular laminin/neurexin/sex hormone-binding (LNS) globulin domains with three interspersed epidermal growth factor (EGF)-like regions

 

Just note the term EGF.

 

In very recent research we see that a reduction in epithelial growth factor may be what is driving some of the key clinical features, such as lack of language.

 

Role of CNTNAP2 in autism manifestation outlines the regulation of signaling between neurons at the synapse

CNTNAP2 has been identified as a master gene in autism manifestation responsible for speech-language delay by impairing the EGF protein domain and downstream cascade. The decrease in EGF is correlated with vital autism symptoms, especially language disabilities.

Autism exhibits genetic heterogeneity, and hence, it becomes difficult to pinpoint one single gene for its manifestation. The gene clusters with varied pathways show the convergence of multiple gene variants, resulting in autism manifestation. Whole-exome sequencing proves to be a reliable tool for deciphering the causal genes for autism manifestation. Deciphering the autism exome identified the mutational landscape derived from single and multi-base DNA variants. Genes carrying mutations were identified in synaptogenesis processes, EGF signaling, and PI3K/MAPK signaling. Protein-protein interactions of NrCAM and CNTN4 with CNTNAP2 increased the impact and burden on autism.

 

 

Shining a light on CNTNAP2: complex functions to complex disorders

TCF4 encodes a basic helix-loop-helix (bHLH) transcription factor that binds near the start site of CNTNAP2 to upregulate its expression (Figure 1a).48 In humans, TCF4 is more highly expressed in the neocortex and hippocampus than in the striatum, thalamus and cerebellum.49 Mutations in TCF4 have been shown to cause Pitt–Hopkins syndrome (PTHS) and three rare TCF4 SNPs are associated with schizophrenia.17^, 49^, 50^, 51 PTHS is characterised by severe intellectual disability, absent or severely impaired speech, characteristic facial features and epilepsy.52 Many of these features are shared with patients carrying CNTNAP2 mutations, leading researchers to test patients with PTHS-like features for CNTNAP2 mutations.17 Two mutations affecting the CNTNAP2 locus (one homozygous and one compound heterozygote) were identified in two independent pedigrees (Table 1). This suggested that disruption of the TCF4–CNTNAP2 pathway could be related to intellectual disability, seizures, and/or behavioural abnormalities.

  

One of our readers in Australia recently queried the potential significance of a mutation (an SNP) in CNTNAP2.  Based on the above, it clearly could be very important. 

What is the common link between TCF4, CNTNAP2 and NRXN1? It would seem to be EGF (epidermal growth factor).

It looks quite possible that EGF is disturbed in much broader autism. It appears that inflammation may reduce EGF levels. It is a rather circular argument, but we also know that EGF reduces inflammation.

To sum up people, with autism likely want more EGF and we already knew that they definitely want less inflammation.  

Decreased Epidermal Growth Factor (EGF) Associated with HMGB1 and Increased Hyperactivity in Children with Autism

These results suggest an association between decreased plasma EGF levels and selected symptom severity. We also found a strong correlation between plasma EGF and HMGB1, suggesting inflammation is associated with decreased EGF.

 

ANKRD17 

Finally, we get back to ANKRD17. 

Our reader Mary has already highlighted this recent paper: - 

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphism

 

Dysmorphic facial features of the ANKRD17-related disorder

 

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder.

 

 

Neonatal growth parameters were normal in the majority of individuals (Table S2) but postnatal growth failure was a feature of almost half of the individuals (height < 2 SD in n ¼ 12 and weight < 2 SD in n ¼ 9). One individual with marked growth failure (individual 3, height 3.8 SD) was under treatment with growth hormone (GH), although GH stimulation testing was normal. Feeding difficulties, especially reduced oral intake, were reported at some stage in 11 individuals, 5 of whom required G-tube nutritional supplementation. Postnatal microcephaly (OFC < 2SD) was noted in seven individuals, and macrocephaly in four (one of these individuals, however, also harbored a pathogenic de novo NSD1 variant (GenBank: NM_022455.4, c.2615T>G [p.Leu872*]). Epilepsy was reported in nine individuals (individuals 1, 2, 16, 19, 21, 25, 27, 28, and 33), with an age of onset of under 2 years for five individuals (individuals 1, 2, 16, 19, and 25). Focal seizures with secondary generalization was the most common seizure subtype, present in five individuals (individuals 1, 2, 21, 25, and 27). One individual had Lennox-Gastaut epilepsy (individual 16), one had tonic seizures with head deviation (individual 19), one had mixed myoclonic and tonic-clonic epilepsy (individual 33), and another a mixture of tonic-clonic and absence seizures (individual 28). Seizures were well controlled (less frequent than every 2 years) in five individuals (individuals 2, 21, 25, 28, and 33), all of whom were on three or fewer antiepileptic drugs (AEDs). Moderate control, with seizures every 2–3 months, was reported in individual 1, who was on Valproate monotherapy. Two individuals had refractory epilepsy during at least parts of their disease course—individual 19 who had frequent tonic seizures in infancy that resolved with topiramate monotherapy and individual 16 Table 2. Frequencies of phenotypic characteristics of individuals with ANKRD17 variants Frequency Sex F ¼ 19, M ¼ 15 Growth Height < 2 SD 12/31 Weight < 2 SD 9/30 OFC < 2 SD 7/31 OFC > 2 SD 4/31 Development DD or ID 31/34 severe 7 moderate 12 mild 5 borderline 7 Motor delay 20/29 Speech delaya 29/32 Other ASD, n ¼ 8; ADHD, n ¼ 4 Neurology Epilepsy 9/33 Abnormal EEG 10/23 Brain MRI abnormalities 11/23 Gait or balance abnormalities 9/25 Spasticity or hypertonia 4/26 Other Recurrent infections 11/33 Feeding problems 11/27 Palate abnormalities 3/34 Hypermobility 9/29 Ophthalmological abnormalities 13/23 Miscellaneous Minor digital anomalies 6 Genitourinary abnormalities 5 Pigmentary abnormalities 4 Scoliosis 3 Abnormal bone mineralization 2 Prominent blood vessels 2 ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder a For details see Table S1 The American Journal of Human Genetics 108, 1138–1150, June 3, 2021 1143 who had multiple seizures every day despite three AEDs. Further details of epilepsy phenotype, including previously trialled AEDs, are noted in Table S2. There were four individuals without epilepsy in whom an abnormal EEG was recorded. Other neurological features include poor balance and/or abnormal gait (9/25) and peripheral spasticity (4/26, one of whom one was microcephalic). Neuroimaging abnormalities were identified in 11 of the 23 individuals in whom an MRI was recorded. Abnormalities include decreased white matter volume (individuals 14, 16, and 18), thinning of the corpus callosum (individuals 14 and 19), optic nerve hypoplasia (individuals 18 and 19), a localized hyperintensity (individuals 7 and 31), right temporal sclerosis (individual 16), dilated Virchow-Robin spaces (individual 6), periventricular nodular heterotopia (individual 30), and an arachnoid (individual 24) and pineal cyst (individual 16). Ophthalmological abnormalities were reported in 13/23 individuals. There were nine individuals with recurrent bacterial infections, one with recurrent viral infections, and one individual with recurrent infections that were both viral and bacterial. The source of bacterial infection was primarily the upper and lower respiratory system and the middle ear (nine individuals) and in some cases required hospitalization. Two individuals were on low-dose prophylactic antibiotics for recurrent otitis media or respiratory tract infections. Notably, individual 26 had a history of pseudomonas and methicillin-resistant staphylococcal aureus (MRSA) infection on his toes. Immunology assessments were recorded in five individuals, details of which can be found in Table S2, with no obvious immunodeficiency identified in these individuals. Generalized joint hypermobility was reported in 9/29 individuals. Notably, there were two individuals with cleft palate in the context of Pierre Robin sequence (PRS) and another with cleft lip and palate. Other infrequent features include minor digital anomalies (n ¼ 6), genitourinary abnormalities (n ¼ 5, of whom three had unilateral renal agenesis), abnormal skin pigmentation (n ¼ 4), scoliosis (n ¼ 3), abnormality of bone mineralization (n ¼ 2), and cutaneous prominence of blood vessels (n ¼ 2). Figure 2 shows the facial features of individuals with the ANKRD17-related neurodevelopmental disorder. Key dysmorphic features include a triangular-shaped face found in 10 of the 24 individuals for whom photos were available with a high anterior hairline (19/24), eyes which are either deep-set (5/24) or almond shaped (8/24) with periorbital fullness (6/24), thick nasal alae and flared nostrils (9/24), full cheeks (7/24), and a thin upper lip (12/24). The degree of dysmorphism was variable, with several individuals (particularly individuals 8 and 10) presenting with only subtle dysmorphic characteristics. Persistence of the high anterior hairline, periorbital fullness, and full cheeks into adulthood is demonstrated in individual 12 (age 30 years) and individual 25 (age 34 years). A number of diagnoses had been considered in several individuals prior to the identification of an ANKRD17 variant, including SATB2-associated syndrome (MIM: 612313) in individual 5 who presented with PRS, triangular facies and speech delay, and Floating-Harbour syndrome (MIM: 136140) in individual 9 who presented with marked short stature (height < 3 SD), microcephaly (head circumference < 2.5 SD), dysmorphic features, and borderline ID. This highlights the phenotypic overlap of the ANKRD17-related disorder with a number of other genetic syndromes, notably those with expressive language delay. In our cohort, significant speech delay was reported in most individuals (n ¼ 29) even in those with IQ in the borderline range. The finding that verbal IQ was reduced relative to performance IQ in three of the five individuals for whom deep neuropsychological phenotyping was available adds further evidence to our observation that expressive language is particularly affected in this disorder

  

How were the 34 individuals identified?

In the Table 1 of the paper, we see that the great majority of the children had been identified from WES (whole exome sequencing), a few had WGS (whole genome sequencing) and just one via micro array testing.

They families clearly opted to share their data, in the hope of some researcher finding it useful later, as Chopra, Amiel and Gordon clearly did after a few years later.

  

How do you figure out the DEGs (differentially expressed genes)?

To treat ANKRD17 deficiency (now known as Chopra Amiel Gordon Syndrome) you have a choice.

·        Increase expression of ANKRD17 via gene therapy, or a drug (if that were possible)

·        Treat some of the downstream DEGs (Differentially Expressed Genes)

Mary asked how you could identify the DEGs, given there is only one paper published on Chopra Amiel Gordon Syndrome.

You can start by reviewing everything known about ANKRD17.

A very good place to start is on the GeneCards website.

https://www.genecards.org/cgi-bin/carddisp.pl?gene=ANKRD17

 

Most people will end up having to learn some new words to understand everything on the above website. 

The first thing to note is just how wide ranging are the functions of this gene and this accounts from the wide-ranging problems associated with it.  It even plays a role in dealing with both viral and bacterial infections.

It is particularly upregulated in the fetal brain and that likely leads to the autism/ID related effects.

Protein differential expression in normal tissues from HIPED for ANKRD17 Gene 

This gene is overexpressed in Lung (18.9), Platelet (15.4), Retina (8.4), and Fetal Brain (6.4).

 

We can see that this gene is associated with Chopra Amiel Gordon Syndrome and Non-Specific Syndromic Intellectual Disability.

Quite possibly, Non-Specific Syndromic Intellectual Disability was used as a term because Chopra Amiel Gordon Syndrome did not yet exist.

But is useful to look up Non-Specific Syndromic Intellectual Disability, to see which other genes are listed.  This then tells you much about what can cause ID.  Follow the link below. 

https://www.malacards.org/card/non_specific_syndromic_intellectual_disability

We see a very long list of syndromes and genes.

There are 61 genes listed.

Going back to the Genecards ANKRD17 page, we can see if there are known protein interactions that might result in autism/ID.

 

 

 For even more related genes/proteins you can look here

 https://string-db.org/cgi/network?taskId=b8Tr5wHDZSsf&sessionId=b1SfhFTkjWxd

  

EIF4E2 does look familiar, and I recall a link to Fragile X.  So, I looked it up.

Note that we see both EIF4E and EIF4E2 - Eukaryotic Translation Initiation Factor 4E Family Member 2.  Note that is has a second name, 4EHP. 

EIF4E2 is a version/homolog of EIF4E 

EIF4E2 = 4EHP 

 

The eIF4E homolog 4EHP (eIF4E2) regulates hippocampal long-term depression and impacts social behavior 

Background: The regulation of protein synthesis is a critical step in gene expression, and its dysfunction is implicated in autism spectrum disorder (ASD). The eIF4E homologous protein (4EHP, also termed eIF4E2) binds to the mRNA 5' cap to repress translation. The stability of 4EHP is maintained through physical interaction with GRB10 interacting GYF protein 2 (GIGYF2). Gene-disruptive mutations in GIGYF2 are linked to ASD, but causality is lacking. We hypothesized that GIGYF2 mutations cause ASD by disrupting 4EHP function.

 

4EHP is expressed in excitatory neurons and synaptosomes, and its amount increases during development. 4EHP-eKO mice display exaggerated mGluR-LTD, a phenotype frequently observed in mouse models of ASD. 

 

Conclusions: Together these results demonstrate an important role of 4EHP in regulating hippocampal plasticity and ASD-associated social behaviors, consistent with the link between mutations in GIGYF2 and ASD.

 

The disruption of protein synthesis (mRNA translation or translation) in the brain by genetic perturbations of its regulators constitutes a known underlying etiology for ASD [2, 3]. For most mRNAs, initiation of translation requires binding of initiation factors to their 5′ end at a modified guanine nucleotide (m⁷GpppN, where N is any nucleotide) termed the 5′ cap [4]. The eukaryotic initiation factor (eIF) 4F complex is comprised of the cap binding protein eIF4E, an mRNA helicase eIF4A, and a molecular scaffold eIF4G. Together these proteins facilitate recruitment of the ribosomal 43S preinitiation complex to the mRNA. Overactivity of eIF4E in humans has been implicated in ASD [5, 6] and ASD-like phenotypes in mice [7, 8]. Indeed, disruption of the proteins regulating eIF4E activity, such as fragile X mental retardation protein (FMRP) [9], cytoplasmic FMR1 interacting protein 1 (CYFIP1) [10], and eIF4E-binding protein 2 (4E-BP2) [8, 11, 12], is implicated in ASD. It is therefore necessary to investigate the function of ASD-linked genes that encode for regulators of translation. Whole-genome sequencing of ASD patients has been invaluable in identifying these genes.

 

If you look up the protein interaction for the Fragile X gene (FMRI), you do indeed see EIF4E close by.  FMR1 encodes the fragile X mental retardation protein.

 

This blog is full of ideas regarding treating Fragile X, because there are so many studies of that type of autism.

It is rather mind-boggling that there are still no approved therapies for Fragile X.  The same holds true for Down Syndrome (DS).  This is a recurring story, where it pays to be the early adopter, not one of the passive followers.

  

Leaky ATP from either Mitochondria or Neurons in Fragile X and Autism

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In that post I suggested “Mirapex - a miracle for Fragile X?”

  

In the post below we saw how EIF4E leads to autism, and how FMRP from Fragile-X affects EIF4E.

 

Vasopressin, Oxytocin, the Lateral Septum, Aggression and Social Bonding, Autism gene NLGN3 and MNK inhibitors for reversing Fragile-X and likely more Autism

  

One of the papers below goes further and suggests

“This work uncovers an unexpected convergence between the genetic autism risk factor Nlgn3, translational regulation, oxytocinergic signalling, and social novelty responses”

“We propose that pharmacological inhibition of MNKs may provide a new therapeutic strategy for neurodevelopmental conditions with altered translation homeostasis”

“Our work not only highlights a new class of highly-specific, brain-penetrant MNK inhibitors but also expands their application from fragile X syndrome to a non-syndromic model of ASD”

 

Regarding Fragile X 

“Collectively, this work establishes eFT508 (an MNK inhibitor) as a potential means to reverse deficits associated with FXS.”

  

Conclusion 

My quick look at the subject suggests that, amongst other likely DEGs, the NLGN (neuroligin) genes are quite possibly miss-expressed.

In humans, alterations in genes encoding neuroligins are implicated in autism and other cognitive disorders.

In Genecards the association is with EIF4E2 rather than the EIF4E, which we know affects neuroligin expression. But EIF4E2 is just a version of EIF4E.

These protein interaction maps are not perfect and different sources often come up with slightly different maps.

 

 

What are Neurexins and Neuroligins?

Neurexins and neuroligins are synaptic cell-adhesion molecules that connect pre- and postsynaptic neurons at synapses, they mediate signalling across the synapse, and shape the properties of neural networks by specifying synaptic functions. Neurexins and neuroligins are therefore very important and can be dysfunctional in autism.

It looks like growth signaling is disturbed in Chopra-Amiel-Gordon Syndrome, but it not always in the same way. Both too much and too little growth are possible.

An MRI would not be a bad idea, and measuring the corpus callosum would be helpful. The corpus callosum connects the right and left side of the brain and is the largest white matter structure in the brain, which means lots of myelin should be there.

If it is very narrow, that would tell you something, hopefully it is normal.  You cannot really change its size, but if it lacked myelination that might be something you could affect.   

Positive Correlations between Corpus Callosum Thickness and Intelligence

Trying the cheap and partially effective treatments for fragile X might be helpful.  It is possible that the Fragile X DEGs overlap with the Chopra-Amiel-Gordon Syndrome DEGs.

The following drugs are cheap generics that are helpful, to some extent, in Fragile-X.

·        Metformin

·        Lovastatin

·        Baclofen

 

As the altered E/I balance is present in Fragile X and most autism, it would be worthwhile trying the E/I corrective therapies that exist, in case one is beneficial.  There are different causes of an E/I imbalance, but since there are not many therapies, it is easier to just try them one by one. 

It is also highly likely that common features of autism may be present, such as

·        oxidative stress (NAC)

·        neuroinflammation (numerous therapies)

·        impaired myelination (Clemastine, Ibudilast, NAG) NAG is not the same as NAC, it is N-acetylglucosamine

·        mitochondrial dysfunction (Carnitine, antioxidants, activate PGC-1 alpha via PPAR gamma e.g. with Pioglitazone)

·        folate receptor antibodies (Calcium folinate)

 

If the Corpus Callosum is smaller than it should be, or is demyelinated, you could try high bio-availability curcumin, in addition to the above pro-myelination therapies.

Which ion channel dysfunctions appear in Chopra-Amiel-Gordon Syndrome?  I did not see any clues, but where there is epilepsy, there is very likely going to be an ion channel dysfunction involved.

Genetic Mutations vs Differentially Expressed Genes (DEGs) in Autism

 

Genes make proteins and you need the right amount in the right place

at the right time.

I should start this post by confessing to not having carried out genetic testing on Monty, now aged 18 with autism.  When I did mention this to one autism doctor at a conference, I was surprised by her reply:- “ You did not need to.  Now there’s no point doing it”.

I got lucky and treated at least some of Monty’s Differentially Expressed Genes (DEGs) by approaching the problem from a different direction.

People do often ask me about what diagnostic tests to run and in particular about genetic testing.  In general, people have far too high expectations regarding such tests and assume that there will be definitive answers, leading to effective therapeutic interventions.

I do include an interesting example today where parent power is leading a drive towards an effective therapeutic intervention in one single gene type of autism.  The approach has been to start with the single gene that has the mutation and look downstream at the resulting Differentially Expressed Genes (DEGs). The intervention targets one of the DEGs and not the mutated gene itself.

This is a really important lesson.

It can be possible to repurpose existing drugs to treat DEGs quite cheaply.  Many DEGs encode ion channels and there are very many existing drugs that affect ion channels.

Entirely different types of autism may share some of the same DEGs and so benefit from the same interventions.

 

Genetic Testing 

Genetic testing has not proved to be the holy grail in diagnosing and treating autism, but it remains a worthwhile tool at a population level (i.e. maybe not in your specific case).  What matters most of all are Differentially Expressed Genes (DEGs), which is something different.

A paper was recently published that looked into commercially available genetic testing.  Its conclusion was similar to my belief that you risk getting a “false negative” from these tests, in other words they falsely conclude that there is no genetic basis for the person’s symptoms of autism. 

 

Brief Report: Evaluating the Diagnostic Yield of Commercial Gene Panels in Autism

Autism is a prevalent neurodevelopmental condition, highly heterogenous in both genotype and phenotype. This communication adds to existing discussion of the heterogeneity of clinical sequencing tests, “gene panels”, marketed for application in autism. We evaluate the clinical utility of available gene panels based on existing genetic evidence. We determine that diagnostic yields of these gene panels range from 0.22% to 10.02% and gene selection for the panels is variable in relevance, here measured as percentage overlap with SFARI Gene and ranging from 15.15% to 100%. We conclude that gene panels marketed for use in autism are currently of limited clinical utility, and that sequencing with greater coverage may be more appropriate.

 

To save time and money, the commercial gene panels only test genes that the company defines as autism genes.  There is no approved list of autism genes. 

You have more than 20,000 genes and very many are implicated directly, or indirectly, in autism and its comorbities. To be thorough you need Whole Exome Sequencing (WES), where you check them all.  

There are tiny mutations called SNPs ("snips") which you inherit from your parents; there are more than 300 million known SNPs and most people will carry 4-5 million.  Some SNPs are important but clearly most are not.  Some SNPs are very common and some are very rare. 

Even WES only analyses 2% of your DNA, it does not consider the other 98% which is beyond the exome.  Whole Genome Sequencing (WGS) which looks at 100% of your DNA will be the ideal solution, but at some time in the future.  The interpretation of WES data is often very poor and adding all the extra data from WGS is going to overwhelm most people involved. 

Today we return to the previous theme of treating autism by treating the downstream effects caused by Differentially Expressed Genes (DEGS).

Genetics is very complicated and so people assume that is must be able to provide answers. For a minority of autism current genetics does indeed provide an answer, but for most people it does not.

Early on in this blog I noted so many overlaps between the genes and signaling pathways that drive cancer and autism, that is was clear that to understand autism you probably first have to understand cancer; and who has time to do that!

Some people’s cancer is predictable. Chris Evert, the American former world No. 1 tennis player, announced that she has ovarian cancer.  Her sister had exactly the same cancer.  Examining family history can often yield useful information and it is a lot less expensive that genetic testing.  Most people’s cancer is not so predictable; sure if you expose yourself to known environmental triggers you raise its chances, but much appears to be random.  Cancer, like much autism, is usually a multiple hit process. Multiple events need to occur and you may only need to block one of them to avoid cancer. We saw this with a genetic childhood leukemia that you can prevent with a gut bacteria. 

Learning about Autism from the 3 Steps to Childhood Leukaemia

What is not random in cancer are the Differentially Expressed Genes (DEGs).

We all carry highly beneficial tumor suppressing genes, like the autism/cancer gene PTEN.  You would not want to have a mutation in one of these genes.

What happens in many cancers is that the individual carries two good copies of the gene like PTEN, but the gene is turned off. For example, in many people with prostate cancer, the tumor suppressor gene PTEN is turned off in that specific part of the body.  There is no genetic mutation, but there is a harmful Differentially Expressed Gene (DEG). If you could promptly turn PTEN expression back on, you would suppress the cancer.

Not surprisingly, daily use of drugs that increase PTEN expression is associated with reduced incidence of PTEN associated cancer.  Atorvastatin is one such drug.

 

DEGs are what matter, not simply mutations

 

In many cases genetic mutations are of no clinical relevance, we all carry several on average.  In some cases they are of immediate critical relevance.  In most cases mutations are associated with a chance of something happening, there is no certainty and quite often further hits/events/triggers are required.

A good example is epilepsy. Epilepsy is usually caused by an ion channel dysfunction (sodium, potassium or calcium) that is caused by a defect in the associated gene. Most people are not born with epilepsy, the onset can be many years later.  Some parents of a child with autism/epilepsy carry the same ion channel mutation but remain unaffected. 

 

Follow the DEGs from a known mutation 

There is a vanishingly small amount of intelligent translation of autism science to therapy, or even attempts to do so.  I set out below an example of what can be done.

 

Pitt Hopkins (Haploinsufficiency of TCF4) 

The syndrome is caused by a reduction in Transcription factor 4, due to mutation in the TCF4 gene.  One recently proposed therapy is to repurpose the cheap calcium channel blocker Nicardipine. Follow the rationale below.

 

↓  means down regulated

↑ means up regulated

1.     Gene/Protein TCF4 (Transcription Factor 4) ↓↓↓↓

2.     Genes SCN10a  ↑↑    KCNQ1 ↑↑

3.     Encoding ion channels  Na_v1.8   ↑↑     K_v7.1   ↑↑

4.     Repurpose approved drugs as inhibitors of K_v7.1 and Na_v1.8 

5.     High throughput screen (HTS) of 1280 approved drugs.

6.     The HTS delivered 55 inhibitors of K_v7.1 and 93 inhibitors of Na_v1.8

7.     Repurposing the Calcium Channel Inhibitor Nicardipine as a Na_v1.8 inhibitor 

           

The supporting science: 

Psychiatric Risk Gene Transcription Factor 4 Regulates Intrinsic Excitability of Prefrontal Neurons via Repression of SCN10a and KCNQ1

  

Highlights

•TCF4 loss of function alters the intrinsic excitability of prefrontal neurons 

•TCF4-dependent excitability deficits are rescued by SCN10a and KCNQ1 antagonists 

•TCF4 represses the expression of SCN10a and KCNQ1 ion channels in central neurons 

•SCN10a is a potential therapeutic target for Pitt-Hopkins syndrome

  

Na_v1.8 is a sodium ion channel subtype that in humans is encoded by the SCN10A gene

K_v7.1 (KvLQT1) is a potassium channel protein whose primary subunit in humans is encoded by the KCNQ1 gene.

  

Transcription Factor 4 (TCF4) is a clinically pleiotropic gene associated with schizophrenia and Pitt-Hopkins syndrome (PTHS).  

SNPs in a genomic locus containing TCF4 were among the first to reach genome-wide significance in clinical genome-wide association studies (GWAS) for schizophrenia  These neuropsychiatric disorders are each characterized by prominent cognitive deficits, which suggest not only genetic overlap between these disorders but a potentially overlapping pathophysiology.

We propose that these intrinsic excitability phenotypes may underlie some aspects of pathophysiology observed in PTHS and schizophrenia and identify potential ion channel therapeutic targets.

Given that TCF4 dominant-negative or haploinsufficiency results in PTHS, a syndrome with much more profound neurodevelopmental deficits than those observed in schizophrenia, the mechanism of schizophrenia risk associated with TCF4 is presumably due to less extreme alterations in TCF4 expression at some unknown time point in development

The pathological expression of these peripheral ion channels in the CNS may create a unique opportunity to target these channels with therapeutic agents without producing unwanted off-target effects on normal neuronal physiology, and we speculate that targeting these ion channels may ameliorate cognitive deficits observed in PTHS and potentially schizophrenia.

 

 

Disordered breathing in a Pitt-Hopkins syndrome model involves Phox2b-expressing parafacial neurons and aberrant Nav1.8 expression

Pitt-Hopkins syndrome (PTHS) is a rare autism spectrum-like disorder characterized by intellectual disability, developmental delays, and breathing problems involving episodes of hyperventilation followed by apnea. PTHS is caused by functional haploinsufficiency of the gene encoding transcription factor 4 (Tcf4). Despite the severity of this disease, mechanisms contributing to PTHS behavioral abnormalities are not well understood. Here, we show that a Tcf4 truncation (Tcf4^tr/+) mouse model of PTHS exhibits breathing problems similar to PTHS patients. This behavioral deficit is associated with selective loss of putative expiratory parafacial neurons and compromised function of neurons in the retrotrapezoid nucleus that regulate breathing in response to tissue CO₂/H⁺. We also show that central Nav1.8 channels can be targeted pharmacologically to improve respiratory function at the cellular and behavioral levels in Tcf4^tr/+ mice, thus establishing Nav1.8 as a high priority target with therapeutic potential in PTHS. 

 

Repurposing Approved Drugs as Inhibitors of K_v7.1 and Na_v1.8 To Treat Pitt Hopkins Syndrome

Purpose:

Pitt Hopkins Syndrome (PTHS) is a rare genetic disorder caused by mutations of a specific gene, transcription factor 4 (TCF4), located on chromosome 18. PTHS results in individuals that have moderate to severe intellectual disability, with most exhibiting psychomotor delay. PTHS also exhibits features of autistic spectrum disorders, which are characterized by the impaired ability to communicate and socialize. PTHS is comorbid with a higher prevalence of epileptic seizures which can be present from birth or which commonly develop in childhood. Attenuated or absent TCF4 expression results in increased translation of peripheral ion channels K_v7.1 and Na_v1.8 which triggers an increase in after-hyperpolarization and altered firing properties.

Methods:

We now describe a high throughput screen (HTS) of 1280 approved drugs and machine learning models developed from this data. The ion channels were expressed in either CHO (K_V7.1) or HEK293 (Na_v1.8) cells and the HTS used either ⁸⁶Rb⁺ efflux (K_V7.1) or a FLIPR assay (Na_v1.8).

Results:

The HTS delivered 55 inhibitors of K_v7.1 (4.2% hit rate) and 93 inhibitors of Na_v1.8 (7.2% hit rate) at a screening concentration of 10 μM. These datasets also enabled us to generate and validate Bayesian machine learning models for these ion channels. We also describe a structure activity relationship for several dihydropyridine compounds as inhibitors of Na_v1.8.

Conclusions:

This work could lead to the potential repurposing of nicardipine or other dihydropyridine calcium channel antagonists as potential treatments for PTHS acting via Na_v1.8, as there are currently no approved treatments for this rare disorder.

  

Repurposing the Dihydropyridine Calcium Channel Inhibitor Nicardipine as a Na_v1.8 inhibitor in vivo for Pitt Hopkins Syndrome

Individuals with the rare genetic disorder Pitt Hopkins Syndrome (PTHS) do not have sufficient expression of the transcription factor 4 (TCF4) which is located on chromosome 18. TCF4 is a basic helix-loop-helix E protein that is critical for the normal development of the nervous system and the brain in humans. PTHS patients lacking sufficient TCF4 frequently display gastrointestinal issues, intellectual disability and breathing problems. PTHS patients also commonly do not speak and display distinctive facial features and seizures. Recent research has proposed that decreased TCF4 expression can lead to the increased translation of the sodium channel Na_v1.8. This in turn results in increased after-hyperpolarization as well as altered firing properties. We have recently identified an FDA approved dihydropyridine calcium antagonist nicardipine used to treat angina, which inhibited Na_v1.8 through a drug repurposing screen.

 

All of the above was a parent driven process.  Well done, Audrey!

Questions remain.

Is Nicardipine actually beneficial to people with Pitt Hopkins Syndrome? Does it matter at what age therapy is started? What about the K_v7.1 inhibitor?

 

Conclusion 

Genetics is complicated, ion channel dysfunctions are complicated; but just a superficial understanding can take you a long way to understand autism, epilepsy and many other health issues.

There is a great deal in this blog about channelopathies/ion channel dysfunctions.

https://epiphanyasd.blogspot.com/search/label/Channelopathy

Almost everyone with autism has one or more channelopathies. Most channelopathies are potentially treatable.

Parents of children with rare single gene autisms should get organized and make sure there is basic research into their specific biological condition.  They need to ensure that there is an animal model created and it is then used to screen for existing drugs that may be therapeutic.  I think they also need to advocate for gene therapy to be developed.  This all takes years, but the sooner you start, the sooner you will make an impact.

Very likely, therapies developed for some single gene autisms will be applicable more broadly.  A good example may be the IGF-1 derivative Trofinetide, for girls with Rett Syndrome. IGF-1 (Insulin-like growth factor 1) is an important growth factor that is required for proper brain development. In the brain, IGF-1 is broken down into a protein fragment called glypromate (GPE). Trofinetide is an orally available version of GPE.

The MeCP2 protein controls the expression of several genes, such as Insulin-like Growth Factor 1 (IGF1), brain-derived neurotrophic factor (BDNF) and N-methyl-D-aspartate (NMDA).  All three are implicated in broader autism. 

https://rettsyndromenews.com/trofinetide-nnz-2566/

In girls with Rett Syndrome the genetic mutation is in the gene MeCP2, but one of the key DEGs (differentially expressed genes) is the FXYD1; it is over-expressed. IGF-1 supresses the activity of FXYD1 and hopefully so does Trofinetide.  Not so complicated, after all!

Medicine is often driven by the imperative to do no harm.

In otherwise severely impaired people, perhaps the imperative should be to try and do some good.

In medicine, time is of the essence; doctors in the ER can be heard to say "Stat!", from the Latin word for immediately, statim.  

How about some urgency in translating autism science into therapy? But then, what's the hurry? Why rock the boat?

On an individual basis, much is already possible, but you will have to do most of the work yourself - clearly a step too far for most people.